The skin is a continually renewing tissue consisting of a large population of transit amplifying (TA) cells with a limited proliferative potential, and a smaller population of keratinocyte stem cells (KSCs) that have a high proliferative potential and are clonogenic. KSCs renew the stem cell population and give rise to TA cells, which are displaced to the suprabasal layers and are lost by terminal differentiation. In the skin, the stem cell population resides in the hair follicle bulge, which is located in the permanent portion of the hair follicle and is protected from both physical damage and the changes the hair follicle undergoes as it cycles from resting (telogen) to active growth (anagen). In the well-known two- stage murine epidermal carcinogenesis model, it is a widely held belief that KSCs are the primary carcinogen target cells (i.e., latent neoplastic cells). A key factor supporting this belief is the fact that DMBA-initiated mice will develop skin tumors upon exposure to a tumor promoter such as TPA whether it is applied a week or a year after initiation. Given that the keratinocyte population renews itself every 6 days in the mouse, the fact that initiated cells persist suggests that these must be slowly cycling cells located in a protected microenvironment. Our current focus is on identification and characterization of the cells that give rise to cutaneous neoplasms in the mouse. As part of this objective, we have investigated the hematopoietic stem and progenitor cell marker, CD34, in the skin and using this marker in combination with alpha-6 integrin and fluorescence activated cell sorting (FACS), have shown that CD34 specifically marks hair follicle bulge keratinocytes, and facilitates isolation of live follicular bulge keratinocytes that represent a subset of alpha-6 integrin bright cells that are quiescent (i.e., predominantly in G1/G0). This work represents the first use of a bulge-specific cell surface marker for physical enrichment of live keratinocyte stem and progenitor cells. The use of the vital dye Hoechst 33342 in the bone marrow and other tissues, including the skin, has identified a small population of cells (called Side Population, or SP cells) that are potentially very early, primitive stem/progenitor cells. We have developed this assay in our lab and are currently characterizing and investigating the proliferative potential of this population, both as progenitor and carcinogen target cells. A comparison was made between CD34+ keratinocytes harvested from either TPA-treated or untreated Tg.AC mice to investigate differential gene expression patterns following tumor promotion. Using nylon cDNA arrays from Clontech probed with PCR-based SMART-amplified cDNA prepared from CD34+ cells isolated from TPA-treated or untreated skin, eleven genes were identified whose expression changed significantly in response to treatment with TPA. Of particular interest was Deleted in Split Hand/Split Foot 1 (Dss1), which is associated with a heterogeneous limb developmental disorder. Overexpression of Dss1 and NDPK-B was detected by RT-PCR and Northern analysis in TPA treated skin (non-tumor bearing; compared to low levels in untreated skin), as well as in cutaneous tumors, including papillomas, squamous cell carcinomas, and spindle cell tumors. Functional studies revealed an increase in foci-forming activity and proliferation of preneoplastic epidermal cells constitutively expressing Dss1. Interestingly, Dss1 induced transformation of stably transfected JB6 epidermal cells was abrogated by addition of a protein kinase C (PKC) specific inhibitor, implicating a possible PKC regulatory role in Dss1 expression. Taken, together, these results suggest that Dss1 is a TPA-inducible gene that may play an important role in the early stages of skin carcinogenesis. In addition, recent studies have implicated a potential involvement of NDBK-B in early-stage neoplastic development in chemically-induced skin carcinogenesis. To assess further the role of hair follicle in cutaneous tumor development, we have utilized the technique of epidermal abrasion, in which the interfollicular epidermis is physically removed. The resulting epidermal regeneration is derived from keratinocytes migrating out from the underlying hair follicles. Any tumors that develop in DMBA-initated wild type or genetically initiated Tg.AC must come from the hair follicle, providing a direct method of investigating the follicular origin of tumors. Tg.AC mice subjected to a single abrasion develop benign papillomas at a similar latency and multiplicity as TPA-treated controls. We have initiated studies in which abraded Tg.AC were compared to similarly abraded, age-matched, FVB/N (parental strain) mice at days 3, 5, 9, and 18 post-abrasion to develop gene expression profiles using high density microarray analysis. The primary goal of these studies is to gain insight into genes that are differentially regulated by the ras transgene and contribute to tumor development. We are exploring the relationship between Ha-ras and p19ARF in benign and malignant tumor development using a bigenic made by crossing p19ARF-null and Tg.AC mice. Tumor studies should shed light on the Ha-ras to p19ARF signaling pathway, thus providing valuable insight into two genes which are stongly linked to numerous mouse and human cancers.